IL18 Gene Polymorphism Is Associated with Total IgE in Adult Subjects with Asthma

The allergic asthma phenotype is characterized by a T helper type 2 (Th2) immune response, based on Immunoglobulin E (IgE)-mediated type 1 hypersensitivity reactions. Total IgE is the sum of all IgE types produced by the human body and is used as a biomarker of inflammation in asthma. We analysed data collected in 143 asthma cases (median age 42.1 years) from the general Italian population (GEIRD survey; 2008–2010) to identify single nucleotide polymorphisms (SNPs) in candidate genes that are associated with total IgE in adult subjects with asthma. These patients reported respiratory symptoms in response to perennial allergens and provided data on 166 SNPs tagging 50 candidate genes or gene regions. Replication of the statistically significant results was performed in 842 asthma cases from other European countries (ECRHS II survey; 1998–2002). SNP rs549908 in interleukin 18 (IL18) gene was significantly associated with total IgE in GEIRD, and this result was replicated in ECRHS II. SNP rs1063320 in the human leukocyte antigen G (HLA-G) gene was identified in GEIRD, but this association was not replicated in ECRHS II. Further investigating IL18 and its biological pathways could be important for developing new therapeutic targets, due to its involvement in inflammatory response processes.


Introduction
Asthma is a multifactorial respiratory disease that depends on the interaction among multiple genetic, environmental, and lifestyle factors [1] and is strongly linked to atopy, allergy, and the response to corticosteroids [2,3]. The allergic asthma phenotype is characterized by a T helper type 2 (Th2) immune response, based on Immunoglobulin E (IgE)-mediated type 1 hypersensitivity reactions [4,5].
IgE belongs to the Ig family, which includes proteins that bind to pathogens and harmful substances (bacteria, viruses, and allergens) to protect the human body [4]. IgE antibodies are produced by activated B cells in response to interleukin (IL) 4 and IL13 cytokines, released by activated Th2 cells. IgE antibodies are released into the circulation and bind effector cells through their receptors [6]. Upon re-exposure to an allergen, the antigen-IgE complex triggers the release of pro-inflammatory mediators (histamine, leukotrienes, and cytokines) by eosinophils, basophils, and mast cells, resulting in local pathophysiological events (such as bronchoconstriction, vasodilation, and airway mucus secretion) that are responsible for triggering respiratory symptoms [5][6][7][8]. Total IgE is the sum of all IgE types that are produced by the human body and is used as a biomarker of asthma inflammation [9,10], with higher levels during childhood and in male individuals [11,12]. Tobacco smoking [13], alcohol consumption [14], and the immunological status of patients [15] also increase total IgE. Associations between total IgE and single nucleotide polymorphisms (SNPs) in some genes, such as Fc epsilon receptor 1A (FCER1A), IL13, signal transducer and activator of transcription 6 (STAT6), cytotoxic T lymphocyte associated protein 4 (CTLA4), and A disintegrin and metalloproteinase domain 33 (ADAM33), have previously been described [16][17][18][19][20][21]. Variants in these genes can lead to dysregulations in the IgE production pathway.
The aim of this study is to identify SNPs in candidate genes associated with total IgE in adult subjects with asthma. This genetic association analysis was performed using Italian data from the Gene Environment Interactions in Respiratory Diseases (GEIRD) study on SNPs in candidate genes [22]. Replication of statistically significant results was carried out within the European Community Respiratory Health Survey (ECRHS) II (www.ecrhs.org (accessed on 18 May 2023)) [23,24].

The GEIRD Survey
GEIRD is a candidate gene-based, (multi)case-control study on the role of genetic and modifiable factors in respiratory diseases (asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and allergic rhinitis) in adults. The protocol is described elsewhere [22]. Briefly, cases and controls were identified through a two-stage process in preexisting cohorts [23][24][25] and in new random samples of the Italian general population [22]. Stage 1 (2007Stage 1 ( -2010 was the screening phase, in which 7413 subjects from the Verona centre received a respiratory health screening questionnaire by mail (response rate 70.7%; Figure 1). Stage 2 (2008-2010) was the clinical phase, in which 2617 responders to the screening questionnaire in Verona underwent a detailed interview, spirometry, and laboratory tests for identifying cases and controls.
Blood samples were collected and stored for IgE titration and genomic DNA extraction according to standardised international protocols [22]. Total IgE levels were measured using the Pharmacia CAP system (Uppsala, Sweden) and were expressed as kiloUnits per Litre (kU/L). Three hundred and eighty-four SNPs tagging 53 candidate genes or gene regions (listed in Table S1) were evaluated in the original survey (2008-2010) [26]. Gene selection in GEIRD is described in the online Supplementary Materials. GoldenGate Genotyping assay (Illumina) was used for genotyping polymorphisms.
In Verona, 1322 cases and controls were identified in the clinical stage, and 997 of these individuals were genotyped. The GEIRD study in this centre was approved by the local ethics committee ("Comitato Etico per la Sperimentazione dell'Azienda Ospedaliera Istituti Ospitalieri di Verona"), and the participants gave their written informed consent after a full explanation of all the aspects of the research project.

Identification of Cases and Controls in GEIRD
Asthma cases were the subjects who met at least one of the following criteria [22]: 1. having reported asthma at any time; 2.
having reported asthma-like symptoms (asthma attacks, wheezing, chest tightness, shortness of breath (SoB) at rest, SoB at night-time, SoB following strenuous activities) or the utilization of anti-asthmatic drugs in the previous 12 months, and having at least one of the following clinical characteristics: Positive methacholine challenge test (provocative dose (PD 20 ) causing a 20% fall in forced expiratory volume in one second (FEV 1 ) < 1 mg); b. Pre-bronchodilator (BD) airflow obstruction (AO) (FEV 1 /forced vital capacity (FVC) <lower limit of normal (LLN) [27] or <70%) and a positive reversibility test (increase in post-BD FEV 1 > 12% and >200 mL with respect to pre-BD FEV 1 after 400 mcg of salbutamol); c.
J. Clin. Med. 2023, 12, x FOR PEER REVIEW 3 of Figure 1. Selection of the asthma cases included in the genetic association analysis (GEIRD surv Verona centre). GEIRD: Gene Environment Interactions in Respiratory Diseases; COPD: chro obstructive pulmonary disease; IgE: Immunoglobulin E. a Subjects who did not fulfil the criteria cases or controls.

Identification of Cases and Controls in GEIRD
Asthma cases were the subjects who met at least one of the following criteria [22]: 1. having reported asthma at any time; 2. having reported asthma-like symptoms (asthma attacks, wheezing, chest tightne shortness of breath (SoB) at rest, SoB at night-time, SoB following strenuo activities) or the utilization of anti-asthmatic drugs in the previous 12 months, a having at least one of the following clinical characteristics: a. Positive methacholine challenge test (provocative dose (PD20) causing a 20% f in forced expiratory volume in one second (FEV1) < 1 mg); b. Pre-bronchodilator (BD) airflow obstruction (AO) (FEV1/forced vital capac (FVC) <lower limit of normal (LLN) [27] or <70%) and a positive reversibility t (increase in post-BD FEV1 > 12% and >200 mL with respect to pre-BD FEV1 af 400 mcg of salbutamol); c. Pre-but not post-BD AO, and post-BD FEV1 ≥ 80% predicted.
The definition of COPD, chronic bronchitis, or allergic rhinitis is reported elsewh [22]. Controls were the subjects who did not fulfil the criteria for case identification a who had pre-BD FEV1 > 70% predicted and no pre-BD AO. A residual group included t individuals who were neither cases nor controls.

Study Subjects
Genetic data were only available for participants in GEIRD stage 2 from Verona. T study subjects were 143 asthma cases (out of 342 genotyped patients) with total I measurement, who had reported respiratory symptoms in response to perennial allerge The definition of COPD, chronic bronchitis, or allergic rhinitis is reported elsewhere [22]. Controls were the subjects who did not fulfil the criteria for case identification and who had pre-BD FEV 1 > 70% predicted and no pre-BD AO. A residual group included the individuals who were neither cases nor controls.

Study Subjects
Genetic data were only available for participants in GEIRD stage 2 from Verona. The study subjects were 143 asthma cases (out of 342 genotyped patients) with total IgE measurement, who had reported respiratory symptoms in response to perennial allergens and, eventually, also to seasonal allergens ( Figure 1). Data from all genotyped subjects were used for additional SNP quality checks.

Genetic Association Analysis
In this analysis, we considered all the SNPs available in the GEIRD dataset (384 polymorphisms tagging 53 genes or gene regions) and, among these SNPs, we analysed the 166 polymorphisms in 50 genes or gene regions (listed in Table S1) that satisfied the following additional quality checks:
Allele frequencies needed to respect Hardy-Weinberg equilibrium (HWE) in the controls (n = 303). The SNPs that were not available in the control group were excluded from the analysis [28]. p-values for testing deviation from HWE were corrected for the False Discovery Rate (FDR) using the Benjamini-Yekutieli procedure [29]; 5.
Linkage disequilibrium (LD; squared correlation between allelic values at two loci (r 2 ) < 0.8) in the asthma cases included in the study (n = 143) to avoid redundant testing. In case of r 2 ≥ 0.8, the SNP with the highest sample size for the homozygous genotype with lower allele frequency was selected for the analysis.
The association with total IgE was assessed separately for each polymorphism using a quasi-gamma log-link regression model [30]. All regression models had total IgE as the outcome and the SNP (classified according to the additive genetic model, with the homozygous genotype with higher allele frequency as the reference), age, and sex as covariates. The strength of the association was measured through the ratio of expected total IgE between the heterozygous genotype (or the homozygous genotype with lower allele frequency) and the reference. The iterated, reweighted least-squares optimization of the deviance was used to obtain the maximum likelihood estimates, taking extra variability into account.

Replication Analysis
SNPs identified in GEIRD were tested for replication in an independent sample of 842 asthma cases who had participated in the ECRHS II survey (from 16 centres located in Estonia, France, Germany, Norway, Spain, Sweden, Switzerland, and United Kingdom). Briefly, ECRHS is an international, population-based cohort study of respiratory health in subjects aged 20-44 years at the time of recruitment (ECRHS I; 1991-1993) [23]. Each participant was mailed a screening questionnaire (stage 1) and a 20% "random sample" of the responders was invited to undergo a detailed clinical examination (stage 2). An additional "symptomatic sample" of subjects with asthma-like symptoms was recruited at stage 2. The participants in ECRHS I stage 2 were re-examined in 1998-2002 (ECRHS II) [24]. Asthma cases were identified according to comparable phenotype criteria (see the online Supplementary Materials) [24] with those used in GEIRD and provided blood samples for genotyping and IgE titration at ECRHS II. Total IgE levels were measured using the Pharmacia CAP system (Uppsala, Sweden) and expressed as kiloUnits per Litre (kU/L). The genotyping method used to analyse the SNPs was the SNPlexTM platform (Applied Biosystems), used according to the manufacturer's instructions [31].
The association between each SNP (identified in GEIRD) and total IgE was evaluated by a 2-level (subject: level 1 unit; centre: level 2 unit) quasi-gamma log-link regression model, with age and sex as adjustment variables. Multilevel modelling was carried out to take the hierarchical structure of ECRHS II data into account. Replication was defined as an effect estimate in the same direction as in GEIRD and, therefore, one-sided p-values were calculated. Replication p-values were corrected to control the FDR using the Simes procedure [32].
Two sensitivity analyses were carried out for the SNPs that were found to be significantly associated with total IgE after FDR correction in the replication set. The first sensitivity analysis was performed within a restricted ECRHS II sample of 51 asthma cases with respiratory symptoms only in the presence of perennial allergens, to exclude an effect due to seasonal exposure. The second sensitivity analysis was performed within the GEIRD control group (n = 288) to test whether the replicated SNPs were associated with total IgE independently of asthma.
All statistical analyses were carried out using STATA software (release 17; StataCorp, College Station, TX, USA), with the exception of the additional SNP quality checks that were performed using R software (version 4.1.2; The R Foundation for Statistical Computing, Vienna, Austria; "HardyWeinberg" package, cran.r-project.org/web/packages/HardyWeinberg).

Main Characteristics of the Asthma Cases
The 143 asthma cases (female 49.7%, past smoker 22.4%, current smoker 28.7%) who were identified in GEIRD and were included in the genetic association analysis had a median age of 42.1 years and a median BMI of 24.5 (Table 1). Of these patients, 69.2% had nasal allergies, 34.3% had eczema/other skin allergies, and 14.0% had an itchy rash. The median pre-BD FEV 1 % predicted was 95.5, the median FVC % predicted was 101.7, and the median FEV 1 /FVC % predicted was 93.3. The geometric mean of total IgE was 111.5 kU/L. A statistically significant difference in the percentage of asthma cases with nasal allergies (p < 0.001) and in the geometric mean of total IgE (p = 0.036) was observed in GEIRD between the study subjects and the 243 eligible patients excluded from the analysis (Table S3). These cases were not evaluated in our study because they had not been genotyped (44 subjects) or, among those with genetic data, because (i) they lacked information on total IgE, (ii) they had reported no respiratory symptoms in the presence of perennial and seasonal allergens, or (iii) they had reported respiratory symptoms in the presence of seasonal allergens only (199 subjects) (Figure 1).

Genetic Association and Replication Analyses
Thirteen polymorphisms were significantly associated with total IgE in GEIRD (Table 2)   The observed association of SNP rs549908 in IL18 gene with total IgE was replicated in ECRHS II (uncorrected one-sided p-value, p = 0.004; FDR-corrected one-sided p-value, p = 0.054). The result for SNP rs1063320 in human leukocyte antigen G (HLA-G) gene did not reach statistical significance after correcting for multiple testing (uncorrected one-sided p-value, p = 0.031; FDR-corrected one-sided p-value, p = 0.198) ( Table 2).
The main characteristics of the GEIRD patients according to rs549908 genotypes (TT, n = 73; TG, n = 60; GG, n = 9) are described in Table 3. The three groups of asthma cases were similar according to the distribution of all variables apart from pre-BD FEV 1 % predicted (p = 0.034) and total IgE (p = 0.005). The expected value of total IgE (obtained with a quasi-gamma log-link regression model, which included the SNP as a qualitative variable and age and sex as adjustment variables) was 154.5 (95% confidence interval, 95%CI: 116.5, 192.6), 270. 7 (196.8, 344.6), and 174.7 (51.6, 297.7) kU/L among the GEIRD patients with TT, TG, or GG genotypes, respectively. Table 3. Main characteristics of the asthma cases a according to SNP rs549908 (IL18) genotype (GEIRD survey).

Sensitivity Analyses
The estimated effect of SNP rs549908 (IL18) within the ECRHS II restricted sample (ratio of expected total IgE (95%CI): 1.48 (0.95, 2.31)) was in the same direction as that obtained within the whole replication set (Table 2). This result supports the hypothesis that the association observed in the replication sample is not due to a seasonal exposure effect, although statistical significance was not reached due to the small sample size of the restricted group (n = 51). No association was found between this polymorphism and total IgE within the control group in the GEIRD dataset, suggesting that this result appears to be dependent on the disease state of the subjects.

Discussion
The main results of the study are the following: • SNP rs549908 in the IL18 gene was associated with total IgE in two independent samples (GEIRD and ECRHS II) of adult subjects with asthma from the general European population; • SNP rs1063320 in the HLA-G gene was linked with this biomarker of inflammation, although statistical significance was not maintained after correcting for multiple testing in the replication sample (ECRHS II).
In this analysis, the effect allele G of the synonymous variant rs549908 was associated with higher levels of total IgE in both the GEIRD and ECRHS II datasets. Despite the fact that the association between SNP rs549908 and total IgE in asthma or other allergic respiratory conditions has not been evaluated in Caucasians, statistically significant results were obtained in this population for polymorphisms in LD with rs549908 (rs1946519 (r 2 = 0.524), rs1946518 (r 2 = 0.524), rs187238 (r 2 = 0.842), rs360718 (r 2 = 0.842), rs360717 (r 2 = 0.842), and rs360721 (r 2 = 1.0); r 2 was calculated for the population of Northern Europeans from Utah (CEU) using the LDlink web application (ldlink.nih.gov (accessed on 18 May 2023))) [33][34][35]. However, these results are not fully comparable with ours due to differences in the study population and age distribution and have not been replicated in other populations. In addition, other studies have identified SNPs in the IL18 receptor gene cluster and in the IL1 family that are linked to asthma-associated biomarkers (i.e., serum IgE, blood eosinophilic counts) in asthma or other allergic disorders [36][37][38][39][40].
The IL18 gene encodes a pleiotropic pro-inflammatory cytokine that is mainly involved in innate and adaptive immune responses in allergic diseases, such as asthma. Asthma is characterized by a Th2-type airway inflammation with eosinophils, IgE production, and airway hyperresponsiveness [41]. This protein belongs to the IL1 family and is expressed by macrophages, dendritic cells, and epithelial cells, as well as smooth muscle cells [42,43]. Its pleiotropic role is determined by which other cytokines are present in the extracellular environment [43][44][45]. IL18 triggers the synthesis and the subsequent release of vasoactive and inflammatory mediators, such as IL4 and IL13, by basophils and mast cells, which in turn amplify IgE production [46,47]. The production and secretion of Th2 cytokines (IL4 and IL13) by differentiated effector cells can also occur in response to IL18 alone, without stimulation of the IgE-receptor complex [48].
We found that the effect allele G of SNP rs1063320 in the HLA-G gene was associated with lower levels of total IgE, although this result did not reach statistical significance after multiple testing correction in the ECRHS II dataset.
The HLA-G gene encodes a major histocompatibility complex class I protein, which is expressed in bronchial epithelium cells. Although the role of HLA-G in asthma is unclear, this protein interacts with T, B, and natural killer cell receptors to modulate immune responses by blocking and/or inhibiting their functions in both soluble and membranebound isoforms [49,50]. In the literature, two genome-wide association studies have identified the HLA-G gene in relation to total IgE [51,52]. Other studies have shown that elevated levels of soluble HLA-G from the bronchoalveolar lavage fluid are associated with low serum IgE [53] or that serum HLA-G expression levels, which are increased in inflammatory diseases (e.g., asthma and allergic rhinitis), are associated with increased serum levels of specific IgE [50,54,55]. Lower levels of HLA-G may be due to the presence of highly polymorphic sites in the 3 untranslated region, such as rs1063320, that control post-transcriptional HLA-G mRNA stability and miRNA regulation [49,[56][57][58]. Despite the lack of replication in ECRHS II, this gene represents an interesting target for further investigation of its role in asthma and its involvement in IgE production.
A major strength of this study is the replication analysis conducted on a relatively large group of asthma patients from other European countries. Furthermore, accurate phenotyping was performed in both GEIRD [22] and ECRHS II [24] using similar standardised protocols. Finally, total IgE was analysed as a continuous rather than categorical variable, as there is no clear consensus in the literature on the cut-off to be used to subdivide patients according to total IgE levels [12,59,60]. The use of a continuous outcome should increase the probability of identifying polymorphisms, as the use of an incorrect cut-off could hide true associations.
Some caveats should be taken into account when interpreting our results. The number of asthma cases in the genetic association analysis (GEIRD dataset; n = 143) is relatively small. However, the evaluation of asthma cases from the general population allows a wider range of disease phenotypes to be captured than the evaluation of clinically selected patients. Furthermore, the limitation due to the relatively small sample size was partly overcome by replicating the results in a larger independent group of patients (ECRHS II dataset; n = 842). In addition, GEIRD is a candidate gene study with a limited number of genotyped SNPs. Finally, two cohorts of European ancestry were analysed in this study, which limits the generalisability of our results to other populations due to the influence of ethnicity on total IgE [61].

Conclusions
We found that SNP rs549908 in the IL18 gene is associated with total IgE among adult subjects with asthma, selected from the general European population in two independent surveys. Further investigation of IL18 and its biological pathways could be important for the development of new therapeutic targets, as it is mainly involved in inflammatory response processes. We also identified SNP rs1063320 in the HLA-G gene in patients from the general Italian population; this deserves additional examination to better understand its role in the allergic asthma phenotype.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jcm12123963/s1, Table S1: List of the 384 tag-SNPs in 53 candidate genes or gene regions that were measured in the GEIRD survey (2008-2010). The 166 SNPs included in the genetic association analysis are reported; Paragraph S1: Gene selection in the GEIRD study; Table S2: List of the references that were used to select the 53 genes or gene regions in the GEIRD survey (2008-2010); Paragraph S2: Definition of asthma in the ECRHS II study; Table S3: Main characteristics of the asthma cases according to their inclusion in the genetic association analysis (GEIRD survey); Paragraph S3: Supplementary information on the ECRHS II study. Data Availability Statement: Due to data protection reasons, the datasets that were analysed in this study cannot be made publicly available.